Part:BBa_K4604015:Design
piG_01b (tetR_bluB)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 710
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1618
Design Notes
The start codon of the bluB was changed to ATG to act as a start for translation in E. coli. The region downstream of the tetA/R promoter was shortened (in comparison to piG_01a/BBa_K4604016) to counteract the leakiness.
Cloning of piG_01b
Plasmid piG_01a (BBa_K4604016) was used as a template. For the PCR we used the general protocol for the Q5 polymerase with an annealing temperature of 56°C and an elongation time of 5 minutes. A Dpn1 digest was done at 37°C for an hour, the DNA was loaded onto an 1% agarose gel with a DNA ladder, the correct bands were cut out and extracted. The parts were assembled using the Golden Gate cloning method according to the general protocol. A transformation was done and the resulting colonies after an approximate 12-14h incubation time were screened by colony PCR. DNA of potential colonies containing the insert was isolated from overnight cultures (5mL LB-medium, 34 mg/mL chloramphenicol) and sent for sequencing to check for correct insertion and no mutation.
Source
Modified piG_01a (BBa_K4604016) using Gibson Assembly.